Pipetting apparatus with integrated liquid level and/or gas bubble detection

ABSTRACT

A pipetting apparatus ( 1 ) comprises a fluidic space ( 7 ), to which a pressure transducer ( 11 ) with a pressure sensor ( 12 ) is attached with a gas filled space ( 15 ). The fluidic space ( 7 ) is defined by a pipette tip ( 2 ), a first tubing ( 5 ) that connects the pipette tip ( 2 ) to a pump ( 4 ), and an active part ( 6 ) of the pump ( 4 ). The pipetting apparatus ( 1 ) according to the present invention is characterized in that the pipetting apparatus ( 1 ) further comprises an impulse generating means ( 16, 18, 19 ) that is in operative contact with a column ( 10 ) of system liquid ( 8 ) inside the fluidic space ( 7 ). The impulse generating means ( 16, 18, 19 ) is designed to induce a vertical movement in this system liquid column ( 10 ), which results in a pressure variation in the gas filled space ( 15 ) that is pneumatically connected with the fluidic space ( 7 ). This pressure variation—as recorded with the pressure transducer ( 11 ) and as processed by a first data processing unit ( 13 ) during utilization of this pipetting apparatus—is taken as an indicator for the detection of penetration or of quitting of a surface ( 17 ) of a liquid, with an orifice ( 3 ) of the pipette tip ( 2 ), of which liquid an amount is to be aspirated and dispensed. This pressure variation is also taken as an indicator for the detection of the presence or the absence of gas bubbles in the system liquid ( 8 ) contained in the fluidic space ( 7 ) of this pipetting apparatus.

RELATED FIELD OF TECHNOLOGY

The present invention relates to a pipetting apparatus for theaspiration (or uptake) and dispensation (or delivering) of volumes ofliquids, such as liquid samples. A pipetting apparatus of this typecomprises a pipette tip with a pipette orifice and a pump. The pipettetip usually is connected to the pump by a first tubing. An active partof the pump, the first tubing and the pipette tip are defining a fluidicspace. This fluidic space is at least partially filled with a systemliquid, such that a meniscus is formed in the fluidic space at an end ofa substantially continuous system liquid column. The typical pipettingapparatus further comprises a pressure transducer with a pressure sensorand a first data processing unit that is designed to process the datareceived from the pressure transducer. The pressure transducer isconnected to the fluidic space via a connection site. The presentinvention also relates to a method of detecting the surface level of aliquid of which an amount is to be pipetted with such a pipettingapparatus. In addition, the present invention relates to a method ofdetecting the presence of gas bubbles in the system liquid of such apipetting apparatus.

RELATED PRIOR ART

Industries applying biochemical techniques in e.g., pharmaceuticalresearch and clinical diagnostics require systems for the handling ofliquid volumes or liquid samples. Automated systems usually comprise oneor more pipetting apparatus operating on liquid containers situated on aworktable. Such systems often are capable to perform operations on theseliquids or samples, such as optical measurements, pipetting, washing,centrifugation, incubation, and filtration. One or more robots, such asCartesian or polar style robots, may be used for operating on such aworktable surface. These robots can carry liquid containers, such assample tubes or microplates. Robots can also be implemented as roboticsample processors (RSP) such as a pipetter for aspirating and dispensingliquids or as a dispenser for delivering of liquids. A central controlsystem or computer usually controls these systems. The primary advantageof such a system is complete hands free operation. Accordingly, thesesystems can run for hours or days at a time with no human intervention.

From U.S. Pat. Nos 4,675,301 and 4,794,085 as well as U.S. Pat. No.5,723,795 a typical pipetting apparatus with a motor driven pump and apipette tip with a pipette orifice is known. The pipette tip isconnected to the pump by tubing. This pipetting apparatus furthercomprises a pressure transducer that is fluidly connected to the tubingat a site of connection. The motor drive of the pump and the pressuretransducer are electrically connected to a data processing unit thatmonitors the drive of the pump and that processes the data received fromthe pressure transducer. In U.S. Pat. Nos. 4,675,301 and 4,794,085, thepump, the tubing and the pipette tip are reported to be completelyfilled with a gas. One of the drawbacks of this approach lays in thecompressibility of gases, which badly influences or even compromisessensitive and precise detection of pressure changes.

The same fluidic space may be completely filled with a system liquidaccording to U.S. Pat. No. 5,723,795. Drawbacks of this approach includea possible covering of the pressure sensor with components of the systemliquid. In addition, moving the liquid-filled tubing (e.g., when therobot moves the pipette tip down towards the fluid surface at a desiredpipetting position) will create spurious pressure signals due to theinertia of the liquid within the tubing. These signals can be largeenough to render the pressure sensor signal unusable during movement,and may require a pause after movement to allow the spurious signal todissipate before a usable signal is available again. Further, it is tobe noted that the system fluid hydraulically couples any mechanicalnoise or vibrations from the mechanism and structure directly into thesensor.

OBJECTS AND SUMMARY OF THE INVENTION

A first object of the present invention is therefore to suggest analternative pipetting apparatus for the aspiration and dispensation ofvolumes of liquids.

A second object of the present invention is to suggest an alternativemethod of detecting the surface level of a liquid, of which an amount isto be pipetted with a pipetting apparatus.

A third object of the present invention is to suggest an alternativemethod of detecting the presence of gas bubbles in the system liquid ofa pipetting apparatus.

These and even further objects are achieved with the features of theindependent claims attached. Advantageous refinements and additionalfeatures of the present invention result from the dependent claims.

The first object is achieved by the provision of a pipetting apparatus,comprising a pipette tip with a pipette orifice and a pump. The pipettetip is connected to the pump by a first tubing. An active part of thepump, the first tubing and the pipette tip are defining a fluidic spacethat is at least partially filled with a system liquid, such that ameniscus is formed in the fluidic space at an end of a substantiallycontinuous system liquid column. The pipetting apparatus furthercomprises a pressure transducer with a pressure sensor and preferablyalso a data processing unit, designed to process the data received fromthe pressure transducer.

The pressure transducer is connected to the fluidic space via aconnection site. The pipetting apparatus according to the presentinvention is characterized in that the pipetting apparatus 1 furthercomprises an impulse generating means that is in operative contact witha column of system liquid inside the fluidic space. The impulsegenerating means is designed to induce a vertical movement in thissystem liquid column, which results in a pressure variation in the gasfilled space that is pneumatically connected with the fluidic space.

In a first preferred embodiment of the pipetting apparatus, the pressurevariation —as recorded with the pressure transducer and as processed bythe first data processing unit—is indicative for the penetration orquitting of a liquid surface with the pipette orifice.

In a second preferred embodiment of the pipetting apparatus, thepressure variation—as recorded with the pressure transducer and asprocessed by the first data processing unit—is indicative for thepresence or absence of gas bubbles in the system liquid contained in thefluidic space.

The second object is achieved by the provision of a method of detectingthe surface level of a liquid of which an amount is to be pipetted,which method is carried out with a pipetting apparatus according to thefirst preferred embodiment. The method according to the inventioncomprises the steps of:

-   -   (a) Filling the fluidic space (7) at least partially with a        system liquid (8) and forming a substantially continuous system        liquid column (10) within the fluidic space (7);    -   (b) Inducing a vertical movement in this system liquid column        (10) by an impulse generating means (16, 18, 19) that is in        operative contact with the system liquid column (10), thereby        causing a pressure variation in the gas filled space (15) that        is pneumatically connected with the fluidic space (7);    -   (c) Recording the pressure variation in the gas filled space        (15) with the pressure transducer (11) and processing the        recorded data with a first data processing unit (13); and    -   (d) Deciding according to the processed data, whether a liquid        surface (17) had been penetrated or quitted with an orifice (3)        of the pipette tip (2).

The third object is achieved by the provision of a method of detectingthe presence of gas bubbles in the system liquid of a pipettingapparatus, which method is carried out with a pipetting apparatusaccording to the second preferred embodiment. The method according tothe invention comprises the steps of:

-   -   (a) Filling the fluidic space at least partially with a system        liquid and forming a substantially continuous system liquid        column within the fluidic space;    -   (b) Inducing a vertical movement in this system liquid column by        an impulse generating means that is in operative contact with        the system liquid column, thereby causing a pressure variation        in the gas filled space that is pneumatically connected with the        fluidic space;    -   (c) Recording the pressure variation in the gas filled space        with the pressure transducer and processing the recorded data        with a first data processing unit; and    -   (d) Deciding according to the processed data, whether gas        bubbles are present in the system liquid that is within the        fluidic space.

ADVANTAGES PROVIDED BY THE INVENTION

Advantages of the present invention comprise:

-   -   The active part (e.g., the piston) of the pump for moving the        system liquid is in direct contact with the in-compressible        system liquid column. Thus, the working surface of the pump is        displaced from the pump into the tubing or pipette tip, e.g.,        close to the pressure transducer.    -   The sensor of the pressure transducer is dry and free of any        deposits originating from the system liquid.    -   Hydraulic transmission of mechanical vibration from the system        to the pressure sensor is limited due to the damping effect of        the gas filled space in front of the pressure sensor.    -   Even abrupt movements of a pipetting robot that comprises such        an apparatus according to the invention are not disturbing the        sensor signal of the pressure transducer, because the gas in the        gas filled space acts as a damper. Thus spurious pressure        signals caused from system fluid inertia while robot is moving        are eliminated.    -   The gas space in the vicinity of the pressure transducer may be        very small, thus, a minimum of a compressible medium is        separating the system liquid from the sample liquid to be        pipetted.    -   Liquid level detection (LLD) on electrically conductive as well        as on nonconductive liquids can be carried out.    -   The simultaneous addition of the LLD according to the invention        to other LLD techniques, such as capacitive LLD, increases the        safety of the liquid level detection.    -   A pipetting apparatus according to the present invention has        (unlike e.g., capacitive or conductive techniques) the ability        to discriminate between gas-filled bubbles and a true liquid        meniscus.

BRIEF DESCRIPTION OF THE DRAWINGS

The device according to the present invention and/or the methodsaccording to the present invention will be described in greater detailon the basis of schematic and exemplary drawings, without these drawingsrestricting the scope of the present invention. It is shown in:

FIG. 1 a vertical section of a first variant of the pipetting apparatuswith the meniscus located in the tubing;

FIG. 2 a vertical section of a second variant of the pipetting apparatuswith the meniscus located in the tubing;

FIG. 3 a vertical section of a third variant of the pipetting apparatuswith the meniscus located in the pipette tip;

FIG. 4 a vertical section of a fourth variant of the pipetting apparatuswith the meniscus located in the pipette tip;

FIG. 5 an alternative to the first variant of FIG. 1;

FIG. 6 a schematic presentation of selected vertical movements of thesystem liquid column within the fluidic space of the pipettingapparatus:

FIG. 6A shows a continuous bidirectional oscillation movement;

FIG. 6B shows a discontinuous bidirectional oscillation movement;

FIG. 6C single bidirectional pulse movement;

FIG. 6D repeated bidirectional pulse movement;

FIG. 6E single unidirectional downward step movement;

FIG. 6F single unidirectional upward step movement;

FIG. 6G repeated unidirectional downward step movement;

FIG. 6H repeated unidirectional upward step movement;

FIG. 7 a vertical section of a piston type pump with a piezo actuator atthe active surface of the piston:

FIG. 7A Plunger movement;

FIG. 7B Piezo movement;

FIG. 8 a vertical section of a piston type pump with a piezo actuatorthat is part of the tubing:

FIG. 8A Plunger movement;

FIG. 8B Piezo movement;

FIG. 9 partial sections through alternative impulse generating meansthat are independent of a pump, whereas:

FIG. 9A shows an electro-mechanical variant;

FIG. 9B shows a first hydraulic variant;

FIG. 9C shows a second hydraulic variant;

FIG. 9D shows a third hydraulic variant;

FIG. 10 erratic movement of the Z-rod of a robotic sample processor wasapplied for pressure monitored liquid level detection (pLLD);

FIG. 11 oscillation movement of the pump piston of a pipetting apparatuswas applied for pressure monitored liquid level detection (pLLD);

FIG. 12 oscillation movement with a modified pinch valve, according toFIG. 9A was applied for pressure monitored liquid level detection(pLLD);

FIG. 13 pressure measurement in the gas filled space of the pipettingapparatus (pLLD) was applied in combination with capacitive liquid leveldetection (cLLD);

FIG. 14 the substantially continuous system liquid column of thepipetting apparatus has characteristic oscillation frequencies, whichcan be monitored with the implemented pressure transducer and pressuresensor;

FIG. 15 system oscillations, preferably as produced with a electricallycontrolled impulse generation means, are correlated with:

FIG. 15A the absence, or

FIG. 15B the presence of gas or air bubbles in the system liquid.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows a vertical section of a first variant of the pipettingapparatus with the meniscus located in the tubing. This pipettingapparatus 1 comprises a pipette tip 2 with a pipette orifice 3 and apump 4. Conventional pipette tips can be used as one-way disposable tipsor as reusable tips as they are known per se. The pump can be a pistonpump like the “CAVRO XP3000 plus Modular Digital Pump” (Tecan SystemsInc., 2450 Zanker Road, San José, Calif. 95138, USA) or a bellows pumpas likewise known from U.S. Pat. No. 5,638,986.

The pipette tip 2 is connected to the pump 4 by a first tubing 5. Anactive part 6 of the pump 4, which preferably is embodied as a piston ora bellow, the first tubing 5 and the pipette tip 2 define a fluidicspace 7 that is at least partially filled with a system liquid 8. Thisfilling with system liquid is such that a meniscus 9 is formed in thefluidic space 7 at an end of a substantially continuous system liquidcolumn 10. In FIG. 1, the meniscus 9 is shown to be in the first tubing5 and outside of the pipette tip 2.

The pipetting apparatus 1 further comprises a pressure transducer 11with a pressure sensor 12 and preferably also comprises a first dataprocessing unit 13. The data processing unit 13 is designed to processthe data received from the pressure transducer 11.

The pressure transducer 11 is connected to the fluidic space 7 via aconnection site 14. This connection site 14 comprises a gas filled space15, which is pneumatically connected with the fluidic space 7. Thepressure sensor 12 limits the gas filled space 15, because here, thepressure sensor 12 is essentially in line with the first tubing 5. Thegas filled space 15 preferably is filled with air or with a chemicallyinert gas like N₂.

The pipetting apparatus 1 further comprises an impulse generating means16, 18, 19 that preferably is electrically controlled and in operativecontact with the system liquid column 10 inside the fluidic space 7 andthat is designed to induce a vertical movement in this system liquidcolumn 10, which results in a pressure variation in the gas filled space15.

There are two major applications for taking advantage of the pressurevariation in the gas filled space 15:

-   -   (A) The pressure variation—as recorded with the pressure        transducer 11 and as processed by the first data processing unit        13—is indicative for the penetration or quitting of a liquid        surface 17 with the pipette orifice 3. Thus, the detection and        interpretation of the pressure variation in the gas filled space        15 is utilized for detection of a surface of a liquid to be        pipetted. This pressure monitored liquid level detection (pLLD)        is independent from the electric conductivity of the liquid that        is to be aspirated and dispensed.    -   (B) The pressure variation—as recorded with the pressure        transducer 11 and as processed by the first data processing unit        13—is indicative for the presence or absence of gas bubbles in        the system liquid 8 contained in the fluidic space 7.

Such inducing of reciprocal movement is now discussed in view of thefirst major application of the pressure variation in the gas filledspace 15, the detection of a liquid level 17 of a liquid to be pipetted.In the context of the present invention, the terms “liquid level 17”,“liquid surface”, and “phase border between a liquid (sample or otherliquid) and the surrounding atmosphere” is treated and understood assynonyms.

As can be seen in FIG. 1 depicting the first variant of the pipettingapparatus, the meniscus 9 of the system liquid column 10 preferably islocated inside the first tubing 5. In this case, the gas filled space 15is a substantial part 31 of the pipette tip 2 volume and a substantialpart 32 of the tube 5 volume. In this variant, the connection site 14may be located in a wall of the pipette tip 2 (see FIG. 4) or in a wallof the first tubing 5 (see FIG. 1). Here, the connection site 14 islocated between the pipette orifice 3 and the meniscus 9 and thepressure transducer 11 is directly attached to the connection site 14,which is open to a part 33 of the fluidic space 7 that is filled withgas. The pipette tip 2 is one selected from a group comprising, e.g.,disposable single pipette tips and disposable multiple pipette tips aswell as single and multiple pipetting needles.

The advantage of this first embodiment lays in its simple construction,which enables the parallel alignment of a larger number (e.g., 8 or 12)of such pipetters in a robotized sample processing unit (not shown) forexample. If only one pipetter channel is fitted to a robot arm (notshown), it may be sufficient to process the data recorded with thepressure transducer 11 by the first data processing unit 13. Control ofthe drive 22 of the pump may be carried out manually.

If, however an automated pipetter or even a multitude of such automatedpipetters are aligned on a robot arm of a laboratory work station (notshown), it is preferred that a second data processing unit 21 isconnected to the motor drive 22 of the pump 4 and to the first dataprocessing unit 13 in order to monitor this motor drive 22 according tothe pressure variation in the gas filled space 15, as recorded by thepressure transducer 11 and processed by the first data processing unit13. All pipetters can be controlled with the second data processing unit21, which may be the central computer of a laboratory workstation.

FIG. 2 shows a vertical section of a second variant of the pipettingapparatus with the meniscus located in the tubing. In contrast to thefirst variant of FIG. 1, the first tubing 5 here comprises an adapter 23for disposable pipette tips. The first tubing 5 also comprises an innertubing 24 and an outer tubing 25. The outer tubing 25 comprises theconnection site 14. The inner and outer tubing extend coaxially to eachother and define a first coaxial gas space 26 between them. This firstcoaxial gas space 26 is pneumatically connected to a second coaxial gasspace 27 located in a disposable pipette tip fixed to the adapter 23.

The advantage of this second embodiment lays in its complete separationof system liquid 8 and sample liquid inside the pipette tip 2. With thispreferred construction, mixing of system liquid and sample liquid isavoided. Also here it is preferred that a second data processing unit 21is connected to the motor drive 22 of the pump 4 and to the first dataprocessing unit 13 in order to monitor this motor drive 22 according tothe pressure variation in the gas filled space 15, as recorded by thepressure transducer 11 and processed by the first data processing unit13. All pipetter channels can be individually controlled with the seconddata processing unit 21, which is integrated in each individualpipetter. The central computer of a laboratory workstation may achievethe synchronization of all pipetter channels.

The inner tubing 24 may be accomplished in a first embodiment as acontinuous tubing constituted of one single plastic piece of the firsttubing 5 that reaches from the pump 4 to the second coaxial gas space27. This embodiment has the advantage of simple construction and ofabsolutely smooth surfaces along the whole first tubing 5. However, thediameter of such first tubing is to be kept relatively small, in orderto reduce flexibility of the tubing. In a second embodiment, the innertubing 24 may be accomplished as an inelastic, stiff tubing 5′ that isconnected to the first tubing 5, which leads to the pump 4. The innertubing 24′ directly reaches to the second coaxial gas space 27.

In both cases, it is preferred that the adapter 23 for disposablepipette tips comprises at least three distance guides 36 on the innerside, these distance guides 36 fix the central position of the innertube 24, 24′ coaxial to the outer tube 25.

These distance guides 36 are spaced from each other so that gas (andtherefore also pressure variations in the gas) can easily pass from thefirst coaxial gas space 26 to the second coaxial gas space 27.

In order to also serve as a dispenser for volumes larger than a pipettetip volume (particularly when using fixed tips), the pipetting apparatus1 in FIG. 2 comprises a pump 4 with a three-way valve 28, from which thefirst tubing 5 is leading towards the pipette tip 2 and a second tubing29, which is leading to a liquid container 30. With this arrangement,large numbers of volumes of the sample liquid stored in the liquidcontainer 30 can be pumped into the first tubing 5 and delivered at theappropriate positions with the pipetting apparatus 1. Also in this case,where the pipetting apparatus 1 is entirely used as a dispenser, thereis no possible contact of the connection site 14 and the liquid to bedispensed. Thus, pressure monitoring with the pressure transducer 11 isguaranteed. In order to achieve easier attachment of the pressuretransducer 11 to the outer tube 25, an additional tube 34 containing thegas filled space 15 connects the sensor 12 to the connection site 14.

As shown in FIG. 2, the liquid container 30 can also be utilized forstorage of system liquid 8. Thus, the entire tubing 5, 29 and pipettetips 2 can be flushed with system liquid 8 via the three-way valve 28.

FIG. 3 shows a vertical section of a third variant of the pipettingapparatus with the meniscus located in the pipette tip. This variant ischaracterized in that the gas filled space 15 is defined as a volume inan additional tube 34 that connects the sensor 12 to the connection site14 and that is sealed from the fluidic space 7 by a flexible membrane35. According to this layout of pipetting apparatus 1, the entirefluidic space 7 can be filled with a substantially continuous systemliquid column 10. The pressure sensor 12 cannot be covered withcomponents of the system liquid 8, because the gas filled space 15 keepsthe pressure sensor 12 dry. This gas filled space 15 can be of minimalextension so that merely a thin gap lays between the flexible membrane35 and the pressure sensor 12. With this pipetting apparatus 1, acertain volume of sample liquid can be aspirated from a sample liquidcontainer. Such container can be any kind of labware, like sample tubes,wells of microplates, troughs etc. Aspiration can be performed with orwithout an air gap between the system liquid 8 and the sample liquid.

FIG. 4 shows a vertical section of a fourth variant of the pipettingapparatus with the meniscus located in the pipette tip. This pipettingapparatus 1 is similar to the one according to first variant (see FIG.1). However, there are characteristic differences between these variantsas seen from FIG. 4:

-   -   (1) There is an adapter 23 for disposable pipette tips        connecting the pipette tip 2 to the first tubing 5.    -   (2) The connection site 14 is not situated in the tubing 5, it        may be placed in the pipette tip 2 (as shown) or in the adapter        23 (not shown).    -   (3) The pressure transducer 11 with its pressure sensor 12 are        connected to the gas filled substantial part of the pipette        volume by an additional tube 34 that comprises gas filled space        15.    -   (4) A constriction element 19 is integrated into the first        tubing 5. This constriction element 19 may be present as a part        of tubing 5 (see FIGS. 4 and 8B) or it may be placed on the        outside of tubing 5 (see FIG. 9A), having an intimate contact to        the tubing 5. This constriction element is the electrically        controlled impulse generating means 16 that preferably is        accomplished here as a piezo actuator 20 in form of tube.

It is expressly noted here that the preferably electrically controlledimpulse generating means 16 not necessarily have to be physicallyconnected with the pump 4 as it derives from element (4) above. However,synchronization of the impulse generating means 16 with the pump actionsis preferred.

An alternative solution of the attachment of the pressure transducer 11to the pipette tip, when compared with the first variant of FIG. 1 orwith the fourth variant in FIG. 4, is seen in FIG. 5. The adapter 23 fordisposable pipette tips connects the pipette tip 2 to the first tubing 5in that the first tubing 5 is penetrating the adapter 23. In addition,an additional tube 34 also penetrates the adapter 23, which isaccomplished as sealing plug. The pressure transducer 11 with itspressure sensor 12 is connected to the additional tube 34. The meniscus9 (not shown) of the system liquid 8 is located within the first tubing5, thus, the pipette tip is serving as the gas filled space 15, which isextended by the additional tube 34, and which is limited by the pressuresensor 12 and the orifice 3 of an empty pipette tip 2 that touches asurface 17 of a sample liquid to be pipetted. Like in FIG. 1, there isno membrane situated between the pressure sensor 12 and the sample orsystem liquid. Like in FIGS. 3 and 4, the pressure transducer isaccomplished as an integrated circuit chip combined with the pressuresensor 12; thus, minimal construction volume is needed outside the firsttubing 5, which is favorable for the preferred construction of a multipipetter liquid handling system that comprises at least one pipettingapparatus 1 as shown in one of the FIGS. 1 to 5. It is preferred thatsuch a multi pipetter comprises 8 or 12 of these pipetting apparatuses1. Such a liquid handling system preferably further comprises a liquidhandling robot and a control unit (not shown).

There are many possible ways of inducing a pressure variation in the gasfilled space 15 of a pipetting apparatus 1 according to the presentinvention. Simple embodiments comprise vibrating Z-drives of a liquidhandling workstation. These vertical vibrations oscillate the column 10of system liquid 8 inside the first tubing 5, which results in thedesired pressure variation or pressure oscillation in the gas filledspace 15 in front of the pressure sensor 12. Also knocking against thetubing may induce a similar effect. Such embodiments have the advantagethat no or only little changes are necessary in the movement controlsfor the vertical movement of the robotized pipetter arm in order toachieve the desired performance. However, such embodiments entail thedrawback of lack of reproducibility (knocking) or such vibrationmovements with the Z-drive may compromise the lifetime of an apparatusand may also be recognized by the laboratory personnel.

Dependent of how the pressure variation or pressure oscillation in thegas filled space 15 in front of the pressure sensor 12 is produced,there are many preferred pressure variations possible, a selection ofwhich is depicted in FIG. 6. There, a schematic presentation of selectedvertical movements of the system liquid column within the fluidic spaceof the pipetting apparatus is drawn in a time-based diagram. Suchmovements comprise a continuous (FIG. 6A) and a discontinuous (FIG. 6B)bidirectional oscillation movement. Both represent a pendulousness witha different multitude of oscillations. Such movements also comprise asingle (FIG. 6C) and a repeated (FIG. 6D) bidirectional pulse movement.Both represent single pendulous oscillations. The series of which may beof different length. Possible ways of producing such pressure variationscomprise the use of an active element that is able to reciprocally movethe liquid column 10. Such active elements comprise a pump piston (seeFIGS. 1-3), pump bellows (not shown), and constriction elements 19 (seeFIG. 4). Additional reciprocally moving elements are shown in FIG. 9below.

Such movements also comprise step-like unidirectional downward or upwardmovements of the liquid column 10 in form of a large number of singledownward (FIG. 6E) or upward step movements (FIG. 6F). Such movementsfurther comprise step-like unidirectional downward or upward movementsof the liquid column 10 in form of series of small repeated numbers ofsingle downward (FIG. 6G) or upward step movements (FIG. 6H). Possibleways of producing such pressure variations comprise the use of an activeelement that is able to unilaterally move the liquid column 10. Suchactive elements comprise a pump piston (18, see FIGS. 1-3) and pumpbellows (not shown). Preferably, the steps are aspiration steps.Possible ways of producing such pressure variations comprise theapplication of triangular or saw-tooth wave shapes.

Any combinations of these movements are applicable too: In FIGS. 6E to6H, e.g., the pressure variation can be produced in that advancing theliquid column is always followed by a retraction of the liquid column.However, advancing is carried out quick (vertical thick line in thegraph) and retraction is carried out slowly (inclined thin line in thegraph). Preferably, a short rest time (thick horizontal line in thegraph) precedes every quick movement.

Inducing a pressure variation in the gas filled space 15 of a pipettingapparatus 1 according to the present invention can be carried out by apiston (18) or by bellows of a pump. FIG. 7 shows a vertical section ofa piston type pump with a piezo actuator 20 at the active surface of thepiston 18. For aspiration or dispensation, the piston 18 can be movedconventionally (FIG. 7A). For inducing pressure variation in the gasfilled space 15 of a pipetting apparatus 1, the active piezo membrane 20is put into operation (FIG. 7B). FIG. 8 shows a vertical section of apiston type pump with a piezo actuator that is part of the tubing. Foraspiration or dispensation, the piston 18 can be moved conventionally(FIG. 8A). For inducing pressure variation in the gas filled space 15 ofa pipetting apparatus 1, the piezo tube 20 is put into constrictionoperation (FIG. 8B). The impulse generating means according to the FIGS.7 and 8 are mainly applicable for reciprocal movements as depicted inFIGS. 6A-6D.

FIG. 9 shows alternative impulse generating means that are independentof a pump and that can also be utilized for inducing a pressurevariation in the gas filled space 15:

FIG. 9A shows an electro-mechanical variant. Fist tubing 5 is leadthrough a cylinder 37. Inside of the cylinder 37, a piston 38, driven bya solenoid 39 and carrying a wedge 40, is moved in essentiallyperpendicular direction against the closed surface of the first tubing5. This movement reversibly deforms the first tubing 5. The preferredfilling of the cylinder 37 is air. Preferably the wedge 40 is ofresilient plastic material in order not to cut the tubing 5 whenpunching against it. A preferably rigid bottom 41 closes the cylinder onthe side opposite to the piston 38. Instead of a wedge (40), also othergeometries formed from solid-state material, like balls and flat orcurved pistons can be utilized.

FIG. 9B shows a first hydraulic variant. Fist tubing 5 is lead throughthe walls of a cylinder 37. Inside of the cylinder 37, the first tubing5 is cut open. The cylinder is filled with a liquid, like system liquid8 (for a pipetter) or sample liquid (for a dispenser). A piston 38,driven by a solenoid 39, is moved in essentially perpendicular directionagainst the open part of the first tubing 5. This movement induces apressure wave in the liquid of the cylinder 37 as well as in the liquidthat is present in the tubing 5. A preferably rigid bottom 41 closes thecylinder on the side opposite to the piston 38.

FIG. 9C shows a second hydraulic variant. Fist tubing 5 is lead throughthe walls of a cylinder 37. Inside of the cylinder 37, the first tubing5 is cut open. The cylinder is filled with a liquid, like system liquid8 (for a pipetter) or sample liquid (for a dispenser). A passivemembrane 42 closes the cylinder 37 opposite to the bottom 41. It isdriven by a piezo stack 43, which is moved in essentially perpendiculardirection against the open part of the first tubing 5. This movementinduces a pressure wave in the liquid of the cylinder 37 as well as inthe liquid that is present in the tubing 5. The preferably rigid bottom41 closes the cylinder on the side opposite to the membrane 42. Thevolume of the cylinder 37 preferably is larger than the volume of thecut away tubing 5. However, it may be considerably smaller than in thevariants of FIGS. 9A and 9B. In an alternative variant (not shown), themembrane 42 is an active piezo membrane and the piezo stack is notpresent. The cylinder filling of the rear side of the membrane 42preferably is air.

FIG. 9D shows a third hydraulic variant. Fist tubing 5 is lead throughthe walls of a cylinder 37. Inside of the cylinder 37, the first tubing5 is cut open. The cylinder is filled with a liquid, like system liquid8 (for a pipetter) or sample liquid (for a dispenser). A passivemembrane 42 closes the cylinder 37 opposite to the bottom 41. Thecylinder filling of the rear side of the membrane 42 preferably is air.The membrane 42 is driven by a driven by sudden expansion of the airwhen heated with the heater 44. Thus, the membrane 42 is partly moved inessentially perpendicular direction against the open part of the firsttubing 5. This movement induces a pressure wave in the liquid of thecylinder 37 as well as in the liquid that is present in the tubing 5. Apreferably rigid bottom 41 closes the cylinder on the side opposite tothe piston 38. The volume of the cylinder 37 preferably is larger thanthe volume of the cut away tubing 5. However, it may be considerablysmaller than in the variants of FIGS. 9A and 9B.

The impulse generating means according to the FIGS. 9A-9D are mainlyapplicable for reciprocal movements as depicted in FIGS. 6A-6D. Theseimpulse generating means are regarded as constriction elements 19, whichare a part of (see FIG. 9B-9D), or which are acting on (see FIG. 9A) thefirst tubing 5.

The method of detecting the surface 17 of a liquid of which an amount isto be pipetted according to the present invention is carried out with apipetting apparatus 1. This pipetting apparatus 1 comprises a pipettetip 2 with a pipette orifice 3 and a pump 4. The pipette tip 2 isconnected to the pump 4 by a first tubing 5.

An active part 6 of the pump 4, the tubing 5 and the pipette tip 2define a fluidic space 7. The pipetting apparatus 1 further comprises apressure transducer 11 with a pressure sensor 12 and preferably also afirst data processing unit 13, designed to process the data receivedfrom the pressure transducer 11. The pressure transducer 11 is connectedto the fluidic space 7 via a connection site 14. The connection site 14comprises a gas filled space 15 that is pneumatically connected with thefluidic space 7 and that is limited by the pressure sensor 12. The gasfilled space 15 preferably is filled with air or with a chemically inertgas like N₂. The pipetting apparatus 1 further comprises a preferablyelectrically controlled impulse generating means 16, 18, 19 that is inoperative contact with the system liquid column 10 inside the fluidicspace 7.

The inventive method comprises the following steps:

-   -   (a) Filling the fluidic space 7 at least partially with a system        liquid 8 and forming a substantially continuous system liquid        column 10 within the fluidic space 7;    -   (b) Inducing a vertical movement in this system liquid column 10        by an impulse generating means 16, 18, 19 that is in operative        contact with the system liquid column 10, thereby causing a        pressure variation in the gas filled space 15 that is        pneumatically connected with the fluidic space 7;    -   (c) Recording the pressure variation in the gas filled space 15        with the pressure transducer 11 and processing the recorded data        with a first data processing unit 13; and    -   (d) Deciding according to the processed data, whether a liquid        surface 17 had been penetrated or quitted with an orifice 4 of        the pipette tip 2.

This pressure monitored liquid level detection (pLLD) can be carried outby discontinuous oscillating, single pulsing, or single stepping thissystem liquid column 10 of step (b) in between of two steps of movingthe pipette orifice 4 towards the liquid surface 17. This liquid leveldetection can also be carried out by continuous oscillating, repeatedpulsing, or repeated stepping this system liquid column 10 of step (b)during movement of the pipette orifice 4 towards the liquid surface 17.Movement towards the liquid surface 17 can be carried out in order topenetrate or to quit the liquid level 17.

In a first experiment (see FIG. 10), erratic movement of the Z-rod of arobotic sample processor (RSP) was applied for pressure monitored liquidlevel detection (pLLD):

The channels II, IV, VI, and VIII of the RSP were equipped with pressuresensors 12, connected to a first data processing unit 13. In order tomonitor and record the plunger or piston 18 movements of the dilutorpumps 4 of the respective channels II, IV, VI, and VIII, the pumps wereadditionally equipped with linear potentiometers. The Z-movement wascarried out with a modified DC-Servo firmware that moves the Z-rodalternating with two different speeds. The scan rate of the data loggerwas 2000/sec.

The voltage indicated is measured at the gate of the signal amplifier ofthe pressure transducer 11. The voltage indicated in the FIGS. 10 to 14can be converted into pressure differences, as 0.02 V are equal to 1mbar.

The recorded process includes the steps of:

-   -   i) flushing of all adapters 23 for disposable pipette tips 2        with system liquid 8;    -   ii) aspiration of a trailing air gap of 10 μl;    -   iii) pick-up of disposable pipette tips 2 (200 μl, standard,        filtered);    -   iv) movement of the RSP arm with four attached pipetting        apparatuses 1 over a liquid container (trough);    -   v) start oscillation; and    -   vi) Z-movement towards the liquid surface 17.

The record is shown in FIG. 10, where picking up the disposable pipettetip 2 (50) and the detection of the liquid level 17 (52) is clearlyvisible.

In a second experiment (see FIG. 11), oscillation movement of the pumppiston 18 of a pipetting apparatus 1 was applied for pressure monitoredliquid level detection (pLLD):

Equipment and process were the same as in the first experiment, exceptthat a modified firmware was used for controlling the pump piston 18,Z-rod movement was standard. The record is shown in FIG. 11, wherepicking up the disposable pipette tip 2 (50), the detection of theoscillation with the pipette tip 2 in air (51), and the detection of theoscillation with the pipette tip penetrating liquid level 17 (52) isclearly visible. The different oscillation amplitudes help todistinguish between the actual positions (air/liquid) of the pipettetip. The piston movement is similar as discussed with FIG. 6A. Thevoltage indicated is measured at the gate of the signal amplifier of thepressure transducer 11. The actual movement of the pump piston 18 was+/−6 steps (out of a total of 3000 possible piston movement steps).Utilizing this pump piston oscillation with 1000 μl pipette tips 2,three steps are equal to a displacement volume of the system liquidcolumn 10 of 1 μl. Thus, here the system liquid column oscillated byabout +/−2 μl. The oscillation pressure measurements enable thediscrimination whether pipette tips 2 with or without filter areutilized.

If a filtered pipette tip is attached, the pressure oscillationmeasurement (see FIG. 11) results in smaller amplitudes, whichnevertheless are characteristic enough for pressure monitored liquidlevel detection.

In a third experiment (see FIG. 12), oscillation movement with amodified pinch valve, according to FIG. 9A was applied for pressuremonitored liquid level detection (pLLD).

Equipment and process were the same as in the second experiment, exceptthat the firmware for controlling the pump piston 18 and the Z-rodmovement was standard. Instead of oscillating the piston, the solenoidvalve was oscillated with 5 Hz; a 1000 μl pipette tip 2 was used here.The record is shown in FIG. 12, where picking up the disposable pipettetip 2 (50), the detection of the oscillation with the pipette tip 2 inair (51), and the detection of the oscillation with the pipette tippenetrating liquid level 17 (52) is clearly visible. The differentoscillation amplitude and characteristics help to distinguish betweenthe actual positions (air/liquid) of the pipette tip. The pistonmovement is similar as discussed with FIG. 8A. Again a filtered pipettetip was attached. The actuation frequency of 5 Hz (5 strokes of thepinch valve per second) proved to be a good value for pLLD with a 1000μl disposable pipette tip 2.

In a fourth experiment (see FIG. 13), pressure measurement in the gasfilled space 15 of the pipetting apparatus 1 (pLLD) was applied incombination with capacitive liquid level detection (cLLD):

The channel II was equipped with a standard cLLD unit and the channel IVof the RSP was equipped with a pressure sensor 12 according to thepresent invention. In order to monitor and record the plunger or piston18 movements of the dilutor pumps 4 of the respective channels II andIV, the pumps were additionally equipped with linear potentiometers. Themovement of the Z-rod was carried out with a standard firmware. The pumppiston 18 was driven for aspiration of liquid at a slow speed with adefined number of 200 steps per second. As before, the utilized pistonpump was able to perform a total of 3000 piston movement steps. The scanrate of the data logger again was 2000/sec.

The recoded process includes the steps of:

-   -   i) flushing of all adapters 23 for disposable pipette tips 2        with system liquid 8;    -   ii) aspiration of a trailing air gap of 10 μl;    -   iii) pick-up of disposable pipette tips 2 (200 μl or 1000 μl,        standard with or without filter);    -   iv) movement of the RSP arm with two attached pipetting        apparatuses 1 over a liquid container (trough).

With the pipette tip 2 of channel II, capacitive LLD was performed twicein order to have a safe mode with double detection of the liquid level17 with the pipette tip of channel II. The detected height or Z-level ofthe liquid surface 17 was stored in a second data processing unit 21 andthe pipette tip of channel II, which was only used for cLLD here, wasthen retracted to a vertical Z-value for save traveling in horizontal Xand Y directions.

First, only pressure measurement was started (60) with the pipette tip 2in air (see FIG. 13). While the tip 2 of channel IV being in air (60),but close to the liquid surface as detected before, slow aspiration (200pump steps per second=66 μl per second) and a downward movement of thetip was started (61) with the Z-rod of a robotic sample processor (RSP)to which the pipetting apparatus 1 of channel IV was attached. No changeof pressure was recorded at that time. Then, the pipette tip 2 ofchannel IV was moved to the liquid surface 17 and—by penetrating theliquid surface 17 (62), aspiration of liquid started immediately and asignificant pressure drop was recorded. The time allowed for thisaspiration was 40 ms with the 200-μl pipette tip. After this time, thepump piston 18 was brought to a full stop (63). This abrupt stopping thepump piston 18, and therefore also the system liquid column 10, acharacteristic pressure oscillation was recognized (64). Thisoscillation results from the mass moment of inertia of the system liquidcolumn 10 in the fluidic space 7 of the pipetting apparatus 1.

The pipette tip 2 of channel IV was then retracted by 30 steps (65) andthe dilutor was set to position zero. This resulted in a dispensation(66-67) of the previously aspirated liquid and air volumes. If a bubbleat the pipette tip orifice 3 is produced by such a dispense, this isdetectable as a distinct pressure peak (67) at the time of breaking ofthe bubble. If only a braking liquid film is formed at the pipette tiporifice 3, this is recorded as a plateau (68) instead of the peak;sometimes both phenomena occur as seen in FIG. 13. Falling of therecoded pressure to approximately the same level (69) as initiallyrecorded (60) proves that the pipette tip 2 was completely emptied bythis dispense. If the pipette tip 2 is empty again, stopping of the pumppiston 18 (70) produces no pressure change in the gas filled space 15 ofthe pipetting apparatus 1. Following this, the RSP arm was moved to adisposal tray and the pipette tip 2 of channel IV was ejected there.

The same procedure was also carried out using standard pipette tips withor without filter; very similar results have been achieved. Also aslower piston speed of only 15 steps/sec or 3 μl/sec revealed similarand reproducible results. However, a higher piston speed is favored inorder to increase the over all processing speed. Using this procedure,the approaching speed of the pipette tip orifice 2 towards the liquidsurface 17 was 20 mm/sec or 40 mm/sec (see FIG. 13) for pLLD. This iscomparable to the standard approaching speed of about 60 mm/sec utilizedfor standard cLLD.

As earlier pointed out, the impulse generating means 16 according to theFIGS. 7, 8, and 9A-9D are mainly applicable for reciprocal movements ofthe liquid column 10 inside the fluidic space 7 of the pipettingapparatus 1 as depicted in FIGS. 6A-6D. Such inducing of reciprocalmovement is now discussed in view of the second major application of thepressure variation in the gas filled space 15, the detection of gasbubbles in the system liquid 8 contained in the fluidic space 7.

Gas or air, usually present as bubbles in the system liquid 8, wouldaffect pipetting precision and accuracy in an intolerable way. Such gasbubbles however, are often not visible for an operator of a roboticsample processor for liquid handling. On the one hand, these gas bubblesare too small or, on the other hand, they appear on hidden positions. Uptoday, it was only possible to detect the presence and effect of suchgas bubbles by an extended and costly gravimetric quality control.

The liquid system, i.e., the substantially continuous system liquidcolumn 10 of the pipetting apparatus 1 according to the presentinvention, has characteristic oscillation frequencies, which can bemonitored with the implemented pressure transducer 11 and pressuresensor 12 (see FIG. 14): The correct working system has very little orno gas bubbles in the system liquid 8. This makes the system liquidcolumn 10 to be rigid and capable to oscillate with a high frequency.The typical frequency is similar at the beginning (61) and at the end ofevery movement the system liquid column 10 is carrying out (63). Thus,at the start and at the end of every aspiration or dispense procedure,the characteristic oscillation frequency of the system liquid column 10can be detected with the pressure sensor 12 in the gas filled space 15of the pipetting apparatus 1 of the present invention. If one comparesthis pressure oscillation at the end of the aspiration (63), with thepressure oscillation (64) after the full stop (63) of the plunger inFIG. 13, the similarity of the oscillation graph is obvious.

System oscillations, preferably as produced with the electricallycontrolled impulse generation means 16 of the present invention, arecorrelated with the absence (see FIG. 15A) or the presence (see FIG.15B) of gas or air bubbles in the system liquid 8. Frequency limits canbe evaluated in order to determine the tolerable amount of gas bubblesin the system liquid 8, which still allows a reasonably precisepipetting to be carried out. No gravimetric quality control is thennecessary anymore. As can be seen from FIG. 15B, gas bubbles present inthe system liquid 8 turns the system to become softer with a decreasedoscillation frequency. Thus, a system with an oscillation as shown inFIG. 15A, can pass the gravimetric quality control; a system producingan oscillation behavior like it is shown in FIG. 15B, will not pass thatquality control. For an easier comparison of the graphs in FIG. 15, asimilar time window (dashed) is drawn. As estimated from this graphs,the oscillation frequency demonstrated for present gas bubbles is abouthalf the frequency of the bubble free system

The method of detecting the presence of gas bubbles in the system liquid8 of a pipetting apparatus 1 according to the present inventioncomprises the steps of:

-   -   (a) Filling the fluidic space 7 at least partially with a system        liquid 8 and forming a substantially continuous system liquid        column 10 within the fluidic space 7;    -   (b) Inducing a vertical movement in this system liquid column 10        by an impulse generating means 16, 18, 19 that is in operative        contact with the system liquid column 10, thereby causing a        pressure variation in the gas filled space 15 that is        pneumatically connected with the fluidic space 7;    -   (c) Recording the pressure variation in the gas filled space 15        with the pressure transducer 11 and processing the recorded data        with a first data processing unit 13; and    -   (d) Deciding according to the processed data, whether gas        bubbles are present in the system liquid 8 that is within the        fluidic space 7.

Advantages of this method comprise:

-   -   The liquid handling system is permanently controlled in terms of        quality of performance, whereas the known gravimetric quality        control provides only momentary information;    -   Due to the on-line control, pipetting errors caused by gas        bubbles in the system liquid 8 can be prevented.

1. Liquid level detection method for a pipetting apparatus (1) that comprises a fluidic space (7), to which a pressure transducer (11) is attached with a gas filled space (15); the fluidic space (7) being defined by a pipette tip (2), a first tubing (5) that connects the pipette tip (2) to a pump (4), and an active part (6) of the pump (4); wherein the liquid level detection method comprises the steps of: (a) filling the fluidic space (7) at least partially with a system liquid (8) and forming a substantially continuous system liquid column (10) within the fluidic space (7); (b) inducing a vertical movement in this system liquid column (10) by an impulse generating means (16, 18, 19) that is in operative contact with the system liquid column (10), thereby causing a pressure variation in the gas filled space (15) that is pneumatically connected with the fluidic space (7); (c) recording the pressure variation in the gas filled space (15) with the pressure transducer (11) and processing the recorded data with a first data processing unit (13); and (d) deciding according to the processed data, whether a liquid surface (17) had been penetrated or quitted with an orifice (4) of the pipette tip (2).
 2. The method of claim 1, wherein the pressure variation—as recorded with the pressure transducer (11) and as processed by a first data processing unit (13) according to step (c)—is indicative for the presence or absence of a filter in the pipette tip (2).
 3. The method of claim 1, wherein the decision according to step (d) is carried out on the base of a pressure oscillation frequency recorded by the pressure transducer (11) during an initial or final part of an aspiration process.
 4. The method of claim 1, wherein vertically moving this system liquid column (10) in step (b) is a discontinuous or continuous bidirectional oscillation movement, caused by oscillation of a piston (18) or of bellows of the pump (4), or caused by oscillation of a membrane (20,42) that is part of the pump (4) or of a constriction element (19).
 5. The method of claim 4, wherein discontinuous oscillating, single pulsing, or single stepping this system liquid column (10) according to step (b) is carried out in between of two steps of moving the pipette tip orifice (4) towards the liquid surface (17).
 6. The method of claim 4, wherein continuous oscillating, repeated pulsing, or repeated stepping this system liquid column (10) of step (b) is carried out during movement of the pipette tip orifice (4) towards the liquid surface (17).
 7. The method of claim 1, wherein vertically moving this system liquid column (10) in step (b) is a single or repeated bidirectional pulse movement caused by impulses carried out with a piston (18) or with bellows of the pump (4), or caused by impulses carried out with a membrane (20, 42) that is part of the piston (18) or of a constriction element (19).
 8. The method of claim 1, wherein vertically moving this system liquid column (10) in step (b) is a single or repeated unidirectional downward or upward step movement caused by a single or repeated forward or backward movement of a piston (18) or of bellows of the pump (4).
 9. The method according to claim 1 for the aspiration and dispensation of volumes of liquids.
 10. Bubble detection method for a pipetting apparatus (1) that comprises a fluidic space (7) to which a pressure transducer (11) is attached with a gas filled space (15); the fluidic space (7) being defined by a pipette tip (2), a first tubing (5) that connects the pipette tip (2) to a pump (4), and an active part (6) of the pump (4); wherein the bubble detection method comprises the steps of: (a) filling the fluidic space (7) at least partially with a system liquid (8) and forming a substantially continuous system liquid column (10) within the fluidic space (7); (b) inducing a vertical movement in this system liquid column (10) by an impulse generating means (16, 18, 19) that is in operative contact with the system liquid column (10), thereby causing a pressure variation in the gas filled space (15) that is pneumatically connected with the fluidic space (7); (c) recording the pressure variation in the gas filled space (15) with the pressure transducer (11) and processing the recorded data with a first data processing unit (13); and (d) deciding according to the processed data, whether gas bubbles are present in the system liquid (8) that is within the fluidic space (7).
 11. The method according to claim 10 for the aspiration and dispensation of volumes of liquids.
 12. The method of claim 10, wherein the decision according to step (d) is carried out on the base of a pressure oscillation frequency recorded by the pressure transducer (11) during an initial or final part of an aspiration process. 